Project Summary The number of children born to opioid-dependent mothers has increased over 300% in the past two decades. As abstinence from opioids during pregnancy is not recommended, opioid medication replacement therapy, such as methadone, represents the standard of care for pregnant women with opioid use disorder thus perpetuating the birth of these opioid-exposed babies. Previous work has shown that prenatal opioid exposure predisposes for future substance misuse. Given its widespread accessibility, alcohol is one of the most likely addictive sub- stances that these prenatal opioid-exposed children will encounter. Due to the significant morbidity and mortality associated with excessive alcohol drinking, it is critically important to understand how prenatal methadone ex- posure (PME) predisposes these children for future problematic alcohol use and how alcohol interacts with PME to produce differential behavioral responses to alcohol. To elucidate mechanisms related to how PME may pro- duce enhanced alcohol-related behaviors, we developed and validated a mouse model of PME. Our model re- capitulates many clinical features of prenatal opioid exposure, including producing neonatal opioid withdrawal. Using our model, we find that PME increases alcohol drinking only in males, consistent with many clinical and preclinical studies that show that males are more severely impacted by prenatal opioid exposure. Given the role of the dorsal striatum brain region in modulating many aspects of alcohol drinking, we biochemically explored the proteome of the dorsal striatum and found that PME had a greater effect on protein and protein phosphory- lation expression in males than females, consistent with our drinking data. Pathway analyses of our proteomics data implicated glutamate and long-term synaptic depression plasticity (LTD) in the dorsolateral striatum (DLS) as being disrupted by PME. We further discovered that PME reduced dorsal striatal glutamate transmission and disrupted LTD. Recent work from our laboratory demonstrates that alcohol induces glutamatergic synaptic plas- ticity and disrupts LTD at anterior insular cortex inputs to the DLS (AICDLS synapses) in mice, but only in male mice and this was associated with enhanced male alcohol drinking behavior. We reasoned that the male-specific, PME-induced increase in binge-like alcohol consumption may utilize similar mechanisms as the male-specific, alcohol-induced AICDLS synaptic changes that govern excessive alcohol consumption. We hypothesize that PME produces synaptic adaptations exclusively in males that enhance AICDLS glutamatergic transmission that in turn govern the elevated binge-like alcohol consumption seen in male, but not female PME mice. In this project we will use a multidisciplinary approach combining home-cage binge drinking with brain slice electro- physiology, dorsal striatal cell type reporter mice, quantitative synaptic proteomics, ultrastructural expansion mi- croscopy, and wir...